One of the main goals in the development of novel therapeutics for proliferative disorders is to generate selective small molecules that potently inhibit cell cycle progression. Several studies have provided evidence of the critical involvement of cyclin/cdk complexes at specific cell cycle regulatory checkpoints (Morgan, 1997, Annu Rev Cell Dev Biol, 13, 261-91; Sherr, 1996, Science, 274, 1672-7). Progression through the cell cycle is driven by activation and deactivation of cyclin/cdk complexes, which start a fundamental cascade of events leading to DNA replication and chromosomal segregation. Tumor development is closely associated with alteration and deregulation of cdks and their regulators, suggesting that inhibitors (antagonists) of cdks may be useful anticancer therapeutics. Therefore, targeting cdk activity has become an attractive strategy in cancer therapy, since it could potentially create a rationally designed inhibitor of a specific process that leads a cell to malignant transformation. To date, several families of chemical inhibitors targeted against different cdk activities have been described (Gray et al., 1998, Science, 281, 533-8; Losiewicz et al., 1994, Biochem Biophys Res Commun, 201, 589-95) and, for some of them, their anticancer therapeutic potential has been demonstrated in preclinical studies (Dai & Grant, 2004, Curr Oncol Rep, 6, 123-30). Recent attention has been focused on biological molecules, especially peptide antagonists, rather than chemotherapeutic agents, that combine the effectiveness of arresting cellular growth through interaction with important cell cycle checkpoint regulators and the low risk of unexpected adverse reactions, thus improving clinical safety and patient tolerability. Therefore, development of pharmacological small peptide molecules able to inhibit cdk activity could be an alternative mechanism-based therapy of great interest in the treatment of neoplasms or other proliferative disorders.
Cdk2 is known to be active in complex with cyclin E at the G1-S boundary, and in complex with cyclin A during S phase progression (Sherr, 1996, Science, 274, 1672-7). Cyclin-dependent kinase 2 (cdk2) is considered the prototypic cell cycle kinase and plays a crucial role in the regulation of cell cycle progression in mammalian cells (Koff et al., 1992, Science, 257, 1689-94; Ohtsubo et al., 1995, Mol Cell Biol, 15, 2612-24). Cdk2 is necessary to pass the G1 restriction point and to drive cells into DNA replication. This enzyme determines whether a cell will leave its resting phase and enter the S phase, a critical determining point, after which a cell is committed to divide.
Among the target substrates that cdks phosphorylate are the members of the retinoblastoma (Rb) family proteins, which play a pivotal role as negative regulators of cell cycle progression (Claudio et al., 1994, Cancer Res, 54, 5556-60). This family includes the product of the retinoblastoma susceptibility gene, the pRb/p105 protein, and the related p107 and pRb2/130 proteins (Hannon et al., 1993, Genes Dev, 7, 2378-91; Mayol et al., 1993, Oncogene, 8, 2561-6; Paggi et al., 1996, J Cell Biochem, 62, 418-30). They share the ability to recruit chromatin-remodeling enzymes and their best characterized targets are the members of the E2F/DP family of transcription factors, generally referred to as E2F (Weinberg, 1995, Cell, 81, 323-30). Both pRb2/p130 and p107 are able to bind cdk2/cyclins A and E (Claudio et al., 1996, Cancer Res, 56, 2003-8). Overexpression of cdk2 with associated cyclins has been shown in several tumors (Al-Aynati et al., 2004, Clin Cancer Res, 10, 6598-605; Olofsson et al., 2004, Int J. Oncol, 25, 1349-55; Zhu, 2004, Cell Cycle, 3). Furthermore, cdk2 has been recently found to be required for centrosome duplication in mammalian cells (Matsumoto et al., 1999, Curr Biol, 9, 429-32; Matsumoto & Maller, 2004, Science, 306, 885-8) suggesting that inhibition of cdk2 activity would be an effective anti-cancer approach. In addition, cdk2 has rapidly emerged as a potential inhibition target by small molecule drugs, which should eventually lead to the development of effective therapies for proliferative disorders (Andrews et al., 2004, Org Biomol Chem, 2, 2735-41; Dai & Grant, 2004, Curr Oncol Rep, 6, 123-30; Gibbs & Oliff, 1994, Cell, 79, 193-8; Hsu et al., 2004, Life Sci, 75, 2303-16; Senderowicz, 2003, Oncogene, 22, 6609-20; Song et al., 2004, Biochem Biophys Res Commun, 317, 128-32).
Previously, it was demonstrated that pRb2/p130, a member of the retinoblastoma family of proteins, acts during cell growth suppression as an inhibitor of cdk2 activity (De Luca et al., 1997, J Biol Chem, 272, 20971-4). The spacer region of pRb2/p130 has a unique amino acid sequence among the other members of the retinoblastoma family, and it is responsible for this inhibitory effect on cdk2 (U.S. Pat. No. 6,297,357). U.S. Pat. Nos. 5,457,049; 5,532,340; 5,807,681; 5,840,506; and 6,663,856, each of which is herein incorporated by reference in its entirety including any references cited therein, also disclose the nucleic acid and polypeptide sequences of the pRB2/p130 spacer domain. The identification and isolation of further true cdk inhibitor peptides exhibiting growth suppressive activity would be useful for designing treatments for cancer therapy; either as an alternative to or in conjunction with other known therapies.